The broad, long-term objectives of this proposal are to define pathogenic mechanisms in human autoimmune myopathies, with a view to defining pathways of therapeutic relevance in this group of diseases. Several recent studies have identified the cytotoxic lymphocyte granule pathway as a pathway of potential importance in autoimmune myositis. In polymyositis, cytotoxic lymphocyte granules are frequently polarized towards muscle cells, indicating that these lymphocytes are in the active process of degranulation. Furthermore, all autoantigens targeted in polymyositis and dermatomyositis are unified by their susceptibility to efficient cleavage by granzyme B (GrB), generating unique fragments not generated during other forms of cell death. We hypothesize that the cytotoxic lymphocyte granule pathway plays a dual role in inducing muscle cell death and in generating the unique forms of autoantigens, which drive the autoimmune response in autoimmune myositis. The specific aims of the proposal are to (1) Define the mechanisms whereby cytotoxic lymphocytes induce muscle cell dysfunction and death in muscle cells in vitro. This will be accomplished by elucidating the effector pathways downstream of GrB in muscle cells in vitro, and by defining the mechanisms responsible for the prominent inhibition of caspases observed in myoblasts; (2) Define the pathways of muscle cell damage in vivo in affected tissues from patients with autoimmune myositis. The activity of the pathways downstream of GrB demonstrated to be of functional relevance in Aim 1 will be directly interrogated using a set of novel reagents that specifically report on the state and activity of GrB, caspases, calpains, nNOS, and muscle structural and regulatory proteins; (3) Elucidate the role of GrB-mediated cleavage and muscle cell differentiation state on the immunogenicity of muscle antigens in an animal model. These studies will address the role of cleavage by GrB in generating the specific immune response to the myositis-specific autoantigen EF-1alpha and define whether the myoblast is the preferential initiator of autoantibody responses to muscle-specific autoantigens. Taken together, these studies will enhance our understanding of the effector mechanisms that participate in the pathogenesis of autoimmune myositis.